required practical 2

Question 1

what is the aim of this practical?

Answer 1

to prepare stained squashes of cells from plant root tips, and then set up and use an optical microscope to identify the stages of mitosis in these stained squashes and calculate a mitotic index

Question 2

what is the risk assessment for this practical?

Answer 2

hazard/risk/prevention
1% toluidine blue solution and 1M HCl/burns or irritation if chemicals get in eye or on skin/wear eye protection, be careful when transferring chemicals, wear gloves or wash off with cold water
sharp instruments (scalpel, knife, scissors, mounted needle)/cuts to skin/carry safely when walking around, cut away from fingers and replace covers when not in use
glassware (can break easily)/cuts to skin/keep away from edge of desk, crush root tips with thumb gently to avoid smashing coverslip

Question 3

what is the method for this practical?

Answer 3

1. use a Pasteur pipette to half-fill a boiling tube with 1M HCl
2. place the boiling tube in a rack and stand in a 40 degree water bath for 5 minutes
3. transfer a garlic clove to the boiling tube so the roots are submerged in acid
4. incubate for 5 minutes
5. remove the clove from the acid and gently rinse the roots with cold distilled water
6. use scissors to cut off 5mm of root tips and let them fall onto a watch glass
7. add a drop of 1% toluidine blue stain to the roots
8. leave the garlic in the stain at room temp for 2 minutes
9. remove excess stain using tissue and rinse root tips in watch glass
10. use forceps to transfer root tips to a clean microscope slide
11. add a drop of water to root tips and spread them with a mounted needle so they don’t overlap
12. place a coverslip on top
13. gently but firmly press down on the coverslip with your thumb to squash and spread out the root tips

Question 4

what is the method for observing and measuring the root cells?

Answer 4

1. place the sample under a microscope and start viewing with the objective lens on the lowest magnification
2. use the coarse adjustment knob to move the lens down to just above the slide
3. use the fine-adjustment knob to refocus until image is clear
4. count total number of cells and number of cells undergoing mitosis to calculate mitotic index

Question 5

what is mitotic index?

Answer 5

the ratio of cells undergoing mitosis to the total number of cells in a sample

Question 6

how do you calculate mitotic index?

Answer 6

number of cells in field of view undergoing mitosis/ total number of cells in field of view

Question 7

why are root tips used?

Answer 7

they are where meristem tissue of food, meaning there is more mitosis occurring

Question 8

why are several roots used?

Answer 8

so that the different stages of mitosis can be observed

Question 9

why are the root tips placed in acid in a water bath 5 mins?

Answer 9

to destroy the cell-surface membranes of the cells so the stain can enter the cell

Question 10

why are the root tips placed in the stain for two minutes?

Answer 10

so that the nuclei of the cells can be stained so they are visible under the microscope